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Determination of butanilicaine in horse plasma and urine by extractive benzoylation and gas chromatography with a nitrogen-phosphorus detector
of Chronuztogrophy. 237 ( 1982) 344-349 J&e&r !Sckdfk PubLkhing Campy, Amsterdam -- Prmted m Tie Nethcdsds
Jozmal
CHRO_M.M. 14,493
Dateumination of butanilicaine in horse plasma and urine by extractive
benzoylation detector”
and gas chromatography
with
a nitrogen-phosphorus
F_ T_ DELBEKE.’ and hI_ DEBACKERE Lahorarcrium
roar Farmncoiogie
siteit Gaz7 Cas&opIein
en Toxicologic
awn de Huis&rm.
Faculteit Diergeme.skw&.
Riyksmirer-
21, B-9000 Ghent (Belgium)
(ReceivedOctober 15th. 198:)
Butaniiicaine [(2-(butylamino)-N-(2chloro-QmethyIphenyl)-acetamide)] is an amide-type local anaesthetic for veterinary use, with a faster and longer activity than procaine’_ A previously described method’ for the analysis of butanilicaine in urine was based on calorimetry. Earlier studies concerning the metabolism of local anaesrhetics3-5, especially butanihcaine breakdown Is the rat2s6, reveal that this drug is
expected to be extensively metabolized in the horse. Therefore the complicated colorimeric method should lack sensivity for the determination of butanilicaine in horse plasma and urine. Although a gas chromatographic (GC) screening procedure with nitrogen specific detection enabling the detection of eleven iocal anaesthetics in horse urine and plasma was recently published’, this method could not been used for the quantitative determination of butanilicaine owing to peak tailing. in the present study an extractive benzoylation reaction based on the Schotten-Baumann procedure has been utilized for the quantitative determination of butanilicaine in biological rluids
Marerials
Butmilicaine triphosphate and the internal standard 2-(butylamino)-N-(2methyM-chiorophenyl)acetamide. I-ICI were supplied by Hoechst (Frankfurt, G.F.R.). Stock solutions were prepared in double-distiiled water. Pentafhtorobe~oyl chloride (PFBCl) was obtained from Aldrich Europe; trifhroroacetic acid anhydride (TFA4) and pentafhroropropionic acid anhydride (PFPA) were purchased from Pierce. The triethanolamine-cyclohexane extraction solvent (CH-TEA) was prepared by briefly refmxing cyclohexane v&h small amounts oftriethanokunine,cooling andseparatingthetwophass TheammoniumbuEer was a saturated NH&l solution adjusted to pH 9.4 with NHaOH. * Prucntcd prtXy at the 4th Me&xzrme.
ZntmsrioMI
Confzmnce
on the Control
of the Lise of Drugs in Horses,
1981.
w21-%73/@_
tSO2.75
8
1982 Eketier scicntifk Publishing
Gxnpay
NO-D%
345
Aii glassware was s&nixed as described earlier’, and the organic solvents (analytical grade) were fresMy distilled before use. Diiutions were made with a IIamihon -digital diluter/dispenser. _. Gas chromaio~itiphy A Varian 3700 equipped with 63Ni and nitrogen-specific detectors and connected to a Varian CDS 111 integrator was used. The glass cohmm (150 x 0.25 cm I.D.).was packed with 3% OV-7 on Chromosorb W HP, SO-100 mesh. The oven temperature was set at 235°C. The injector and detector temperatures were kept at 250°C and 300°C respectively. Nitrogen (25d/tin) was used as carrier gas. Mass
spectrometry
A mass spectrum was obtained on a HP 5995 apparatus, the column being a 25-m SP-2 100 fused-silica column. Methods
For the determination of butanilicaine in horse plasma OF urine, 2 ml of the ~biological fluid, 50 ti of the aqueous internal standard solution (10 pg/ml) and 0.2 ml of ammonium buffer were mixed with 6 ml CH-TEA and 10 fl of PFBCl (5% in eyclohexane) for 5 min. After a brief centrifugation, 5 ml of the organic phase were transferred to a tapered test-tube and evaporated to dryness under nitrogen at 40°C. The residue was redissobzd in 50 ~1of ethyl acetate, and 1 ~1was injected into the gas chromatograph (nitrogen-phosphorus detection mode)_ A standard graph was prepared by treating known amounts of butanilicaine triphosphate in plasma according to this procedure_ The recoveries were obtained by adding different amounts of butanihcaine triphosphate to 2 ml of plasma OF urine and extractive benzoylation (rotating or vorte.xing) foliowed by the addition of the internal standard as a methanolic solution, evaporation and subsequent reaction with PFBCl. RESULTS
AND
DISCUSSION
Because the gas-liquid chromatographic separation of compounds containing amino groups generally results in peak tailing due to spurious adsorption onto the coiumn, several approaches have been used for the derivatization of the amine function in butanilicaine and the isomeric internal standard. The acylation with TFAA and PFPA was not complete at room temperature and the compounds formed were easily hydrolysed. Therefore benzoylation of the secondary amino group in butaniiicaine was tried. Benzoyl derivatives can be made from alcohols, thiols and amines by treating the residue obtained by benzene extraction with pyridiie and benzoyl chloride and shaking over several hoursg. The Schotten-Baumann procedure. whereby an alkaline glycerol solution was shaken with b9nzoyl chloride, followed by extracting the derivatives, was applied by Decroix et aLEO. The Schotten-Baumann type acylation reaction -was also used for the determination of phenohc amines in water OF plasma”. PFBCl derivatives are generahy made with trimethylamine as catalyst” OF at elevated tcmperatures’3*‘*. IQ this work, however, a Schotten-Baumam reaction was performed
.__.-_ -.
Although t&epent&uorobenzoyl group appearsto be the one that confers the *test sensitivityfor elect_rotipkre detectionof amines’?, this detectionmethod could not be used here. In&d, t.he formation of the strongly ekctron-capturing pentafluorobenzoica+d (PFBH) causes serious GC interference.Although PFBH can be removed by extractingthe orgkic phase with 1 N NaQW6, this procedure could not be used here owing to the hydrolysisof the benzami’deand butanilicaitie itself at this pH_ Nevertheless,the use of the nitrogen-specific‘ detector enables an acceptabledetectionknit to be achieved_ 195
CH,-
238 308
413
_ I
50
100
150
200
250
- 300
Fig 3. Ekctron impact mass spwtmrn of the pentafluorol
350
400
-de of butanilitine
450
-4
NOTES
34s
TABLE
I
RkkOVErUES
OF BUTANKKAiNE
m,,-19s(100.0%)
ADDED
TO HORSE PIAS-MA OR URINE
m,,-¶40(11.3%)
In,,-306(26.3%)
c
=a5 1~1/~-167:17.3%'s)
r CHfN
/COC6F6
1
'C4H!3
rCH,-C%CH,-C-
_: .
m,z’233(34.096)
?
1
CsFs
+
.- -_
Fig_ & I?ostulated mass fiagsnmtation pattern of the N-pentakoro~cie
of butadbk-
The extractivepentz&orobenzoylation of butanihcaineand the internalstand&d was completeafter ca. 5 min at room temperatrrre. The.recoveries(Table f) were nearLyquantitative_ A standardgraph with a linear~onceutrationrangein the intervai5-500 r&ml is presentedin Fig- 1 (r = 0.999, n = 38). With 2 ml of plasma, the detectionlimitwas evaluatedat 5 ng of butanihcainetriphosphate.Fig. 2 shows a typical chromatogram obtaitied by processi ng bkmk and-spiked korse plasma as described in the Experimental section. The mass spectrumof the pentailuorobenzamideof butanihcainedid not give a motecularion (Fig. 3). However, diagnosticions at m/z = 413,308,280,23S and 140 were observed_The postulatedstructuresfor these ions are shown in Fig. 4. CONCLUSIONS
With triethanolamineas catalyst, the extractivebenzoylationunder SchottenBaumannconditionsof butanilicainewith PFBCl and subsequentGC with nitrogenspecificdetectionis a simple, rapid and sensitivetechniquefor the determinationof butanihcainein plasma and urine. ACKNOWLEDGEMENT
The authorsthank Mrs. G. Demey and Mr. N. Dcsmet for technicalassistance. Thanks are also due to Hoechst AG for a gift of butanilicaineand some isomers.This work was supported by the WelIcome Foundation CentenaryGrant. REFERENCES 1 2 3 4 5 6 7 8 9 IO 1I I2 13 14 15 I6
L. A. T. D. E.
Ther. Xuzuqm-Scclun.fe&berg5Arch_, 220 (1953) 300. HaussIer and I- Ther. dr=neim.-borsch., 3 (1953) 609. Tobin, J. W. Bbke, C. Y_ Tai. L Sturma and S. Arnett Amer. L t’et. Rex, 37 (1976) 1165. Courtot. fish rT2r.1.. 3: (1979) 205. Hanson, in P_ L&&at (Editor), fnremarfonai .Ekc_vciopacdikzof Pharmacolog_v and Therapemics. VoL 1, 1971. Section 8. Per_mon. p_ 739. S. K2sai, Uszku C-try .Wed_ J., 9 (1963) 91. F. T. De&eke, M. Debackere and N. Desmet, J_ Cfirunmrugr_, 206 (1981) 594. R V. Smith. Inter. Lab., Nov.-Dec. (1976) 71. L. Gasto and R. &u-rem, Anal. Chim. rfczu, 61 (1972) 253. G. Decroix, J. Gobert and R De Deunmerder, Anrrl Biochem., 25 (1968) 523. G. J. Wright, J. F. L;tng, R. E. !!_emieux2nd h-f. J. Goadftiend, Jr., Drug. Merub. Rev_, 4 (1975) 267. G. R. WiIkinson, Anal. Letr., 3 (1970) 289. K- K. Midha, I. J_ MeGilver2y and J_ K. Cooper, Cum. J. Phtzrm. Sci., 14 (1979) 18. K- Blau. 1. M- Qaxton, G_ Ism&an and M. SmdIer, ,I. Cirromatogr, 163 (1979) 135. A. C. Moffat, E C. Homing,S. B. Metin and M. Rowland, i. Chromafogr_. 66 (1972) 2%. K. Blau 2nd G. King, in K. BIau and G. King (Editors), Handbook of Derigarives for Chromam&aphy, He$.en & Son. London, 1977, p_ 133.
Jozmal
CHRO_M.M. 14,493
Dateumination of butanilicaine in horse plasma and urine by extractive
benzoylation detector”
and gas chromatography
with
a nitrogen-phosphorus
F_ T_ DELBEKE.’ and hI_ DEBACKERE Lahorarcrium
roar Farmncoiogie
siteit Gaz7 Cas&opIein
en Toxicologic
awn de Huis&rm.
Faculteit Diergeme.skw&.
Riyksmirer-
21, B-9000 Ghent (Belgium)
(ReceivedOctober 15th. 198:)
Butaniiicaine [(2-(butylamino)-N-(2chloro-QmethyIphenyl)-acetamide)] is an amide-type local anaesthetic for veterinary use, with a faster and longer activity than procaine’_ A previously described method’ for the analysis of butanilicaine in urine was based on calorimetry. Earlier studies concerning the metabolism of local anaesrhetics3-5, especially butanihcaine breakdown Is the rat2s6, reveal that this drug is
expected to be extensively metabolized in the horse. Therefore the complicated colorimeric method should lack sensivity for the determination of butanilicaine in horse plasma and urine. Although a gas chromatographic (GC) screening procedure with nitrogen specific detection enabling the detection of eleven iocal anaesthetics in horse urine and plasma was recently published’, this method could not been used for the quantitative determination of butanilicaine owing to peak tailing. in the present study an extractive benzoylation reaction based on the Schotten-Baumann procedure has been utilized for the quantitative determination of butanilicaine in biological rluids
Marerials
Butmilicaine triphosphate and the internal standard 2-(butylamino)-N-(2methyM-chiorophenyl)acetamide. I-ICI were supplied by Hoechst (Frankfurt, G.F.R.). Stock solutions were prepared in double-distiiled water. Pentafhtorobe~oyl chloride (PFBCl) was obtained from Aldrich Europe; trifhroroacetic acid anhydride (TFA4) and pentafhroropropionic acid anhydride (PFPA) were purchased from Pierce. The triethanolamine-cyclohexane extraction solvent (CH-TEA) was prepared by briefly refmxing cyclohexane v&h small amounts oftriethanokunine,cooling andseparatingthetwophass TheammoniumbuEer was a saturated NH&l solution adjusted to pH 9.4 with NHaOH. * Prucntcd prtXy at the 4th Me&xzrme.
ZntmsrioMI
Confzmnce
on the Control
of the Lise of Drugs in Horses,
1981.
w21-%73/@_
tSO2.75
8
1982 Eketier scicntifk Publishing
Gxnpay
NO-D%
345
Aii glassware was s&nixed as described earlier’, and the organic solvents (analytical grade) were fresMy distilled before use. Diiutions were made with a IIamihon -digital diluter/dispenser. _. Gas chromaio~itiphy A Varian 3700 equipped with 63Ni and nitrogen-specific detectors and connected to a Varian CDS 111 integrator was used. The glass cohmm (150 x 0.25 cm I.D.).was packed with 3% OV-7 on Chromosorb W HP, SO-100 mesh. The oven temperature was set at 235°C. The injector and detector temperatures were kept at 250°C and 300°C respectively. Nitrogen (25d/tin) was used as carrier gas. Mass
spectrometry
A mass spectrum was obtained on a HP 5995 apparatus, the column being a 25-m SP-2 100 fused-silica column. Methods
For the determination of butanilicaine in horse plasma OF urine, 2 ml of the ~biological fluid, 50 ti of the aqueous internal standard solution (10 pg/ml) and 0.2 ml of ammonium buffer were mixed with 6 ml CH-TEA and 10 fl of PFBCl (5% in eyclohexane) for 5 min. After a brief centrifugation, 5 ml of the organic phase were transferred to a tapered test-tube and evaporated to dryness under nitrogen at 40°C. The residue was redissobzd in 50 ~1of ethyl acetate, and 1 ~1was injected into the gas chromatograph (nitrogen-phosphorus detection mode)_ A standard graph was prepared by treating known amounts of butanilicaine triphosphate in plasma according to this procedure_ The recoveries were obtained by adding different amounts of butanihcaine triphosphate to 2 ml of plasma OF urine and extractive benzoylation (rotating or vorte.xing) foliowed by the addition of the internal standard as a methanolic solution, evaporation and subsequent reaction with PFBCl. RESULTS
AND
DISCUSSION
Because the gas-liquid chromatographic separation of compounds containing amino groups generally results in peak tailing due to spurious adsorption onto the coiumn, several approaches have been used for the derivatization of the amine function in butanilicaine and the isomeric internal standard. The acylation with TFAA and PFPA was not complete at room temperature and the compounds formed were easily hydrolysed. Therefore benzoylation of the secondary amino group in butaniiicaine was tried. Benzoyl derivatives can be made from alcohols, thiols and amines by treating the residue obtained by benzene extraction with pyridiie and benzoyl chloride and shaking over several hoursg. The Schotten-Baumann procedure. whereby an alkaline glycerol solution was shaken with b9nzoyl chloride, followed by extracting the derivatives, was applied by Decroix et aLEO. The Schotten-Baumann type acylation reaction -was also used for the determination of phenohc amines in water OF plasma”. PFBCl derivatives are generahy made with trimethylamine as catalyst” OF at elevated tcmperatures’3*‘*. IQ this work, however, a Schotten-Baumam reaction was performed
.__.-_ -.
Although t&epent&uorobenzoyl group appearsto be the one that confers the *test sensitivityfor elect_rotipkre detectionof amines’?, this detectionmethod could not be used here. In&d, t.he formation of the strongly ekctron-capturing pentafluorobenzoica+d (PFBH) causes serious GC interference.Although PFBH can be removed by extractingthe orgkic phase with 1 N NaQW6, this procedure could not be used here owing to the hydrolysisof the benzami’deand butanilicaitie itself at this pH_ Nevertheless,the use of the nitrogen-specific‘ detector enables an acceptabledetectionknit to be achieved_ 195
CH,-
238 308
413
_ I
50
100
150
200
250
- 300
Fig 3. Ekctron impact mass spwtmrn of the pentafluorol
350
400
-de of butanilitine
450
-4
NOTES
34s
TABLE
I
RkkOVErUES
OF BUTANKKAiNE
m,,-19s(100.0%)
ADDED
TO HORSE PIAS-MA OR URINE
m,,-¶40(11.3%)
In,,-306(26.3%)
c
=a5 1~1/~-167:17.3%'s)
r CHfN
/COC6F6
1
'C4H!3
rCH,-C%CH,-C-
_: .
m,z’233(34.096)
?
1
CsFs
+
.- -_
Fig_ & I?ostulated mass fiagsnmtation pattern of the N-pentakoro~cie
of butadbk-
The extractivepentz&orobenzoylation of butanihcaineand the internalstand&d was completeafter ca. 5 min at room temperatrrre. The.recoveries(Table f) were nearLyquantitative_ A standardgraph with a linear~onceutrationrangein the intervai5-500 r&ml is presentedin Fig- 1 (r = 0.999, n = 38). With 2 ml of plasma, the detectionlimitwas evaluatedat 5 ng of butanihcainetriphosphate.Fig. 2 shows a typical chromatogram obtaitied by processi ng bkmk and-spiked korse plasma as described in the Experimental section. The mass spectrumof the pentailuorobenzamideof butanihcainedid not give a motecularion (Fig. 3). However, diagnosticions at m/z = 413,308,280,23S and 140 were observed_The postulatedstructuresfor these ions are shown in Fig. 4. CONCLUSIONS
With triethanolamineas catalyst, the extractivebenzoylationunder SchottenBaumannconditionsof butanilicainewith PFBCl and subsequentGC with nitrogenspecificdetectionis a simple, rapid and sensitivetechniquefor the determinationof butanihcainein plasma and urine. ACKNOWLEDGEMENT
The authorsthank Mrs. G. Demey and Mr. N. Dcsmet for technicalassistance. Thanks are also due to Hoechst AG for a gift of butanilicaineand some isomers.This work was supported by the WelIcome Foundation CentenaryGrant. REFERENCES 1 2 3 4 5 6 7 8 9 IO 1I I2 13 14 15 I6
L. A. T. D. E.
Ther. Xuzuqm-Scclun.fe&berg5Arch_, 220 (1953) 300. HaussIer and I- Ther. dr=neim.-borsch., 3 (1953) 609. Tobin, J. W. Bbke, C. Y_ Tai. L Sturma and S. Arnett Amer. L t’et. Rex, 37 (1976) 1165. Courtot. fish rT2r.1.. 3: (1979) 205. Hanson, in P_ L&&at (Editor), fnremarfonai .Ekc_vciopacdikzof Pharmacolog_v and Therapemics. VoL 1, 1971. Section 8. Per_mon. p_ 739. S. K2sai, Uszku C-try .Wed_ J., 9 (1963) 91. F. T. De&eke, M. Debackere and N. Desmet, J_ Cfirunmrugr_, 206 (1981) 594. R V. Smith. Inter. Lab., Nov.-Dec. (1976) 71. L. Gasto and R. &u-rem, Anal. Chim. rfczu, 61 (1972) 253. G. Decroix, J. Gobert and R De Deunmerder, Anrrl Biochem., 25 (1968) 523. G. J. Wright, J. F. L;tng, R. E. !!_emieux2nd h-f. J. Goadftiend, Jr., Drug. Merub. Rev_, 4 (1975) 267. G. R. WiIkinson, Anal. Letr., 3 (1970) 289. K- K. Midha, I. J_ MeGilver2y and J_ K. Cooper, Cum. J. Phtzrm. Sci., 14 (1979) 18. K- Blau. 1. M- Qaxton, G_ Ism&an and M. SmdIer, ,I. Cirromatogr, 163 (1979) 135. A. C. Moffat, E C. Homing,S. B. Metin and M. Rowland, i. Chromafogr_. 66 (1972) 2%. K. Blau 2nd G. King, in K. BIau and G. King (Editors), Handbook of Derigarives for Chromam&aphy, He$.en & Son. London, 1977, p_ 133.